Prevention of cancers, non-cancer, hormone dependent diseases and cardiovascular diseases by use of hydroxymatairesinol, and a pharmaceutical preparation, food additive and food product comprising hydroxymatairesinol

ABSTRACT

This invention relates to methods for prevention of cancers, certain non-cancer, hormone dependent diseases and/or cardiovascular diseases in a person, based on administering of hydroxymatairesinol to said person. The invention also concerns a method for increasing the level of enterolactone or another metabolite of hydroxymatairesinol in a person&#39;s serum thereby causing prevention of a cancer or a certain non-cancer, hormone dependent disease in a person, based on administering of hydroxymatairesinol to said person. Furthermore, this invention relates to pharmaceutical preparations, food additives and food products comprising hydroxymatairesinol.

FIELD OF THE INVENTION

[0001] This invention relates to methods for prevention of cancers,certain non-cancer, hormone dependent diseases and/or cardiovasculardiseases in a person, based on administering of hydroxymatairesinol tosaid person. The invention also concerns a method for increasing thelevel of enterolactone or another metabolite of hydroxymatairesinol in aperson's serum thereby causing prevention of a cancer or a certainnon-cancer, hormone dependent disease in a person, based onadministering of hydroxymatairesinol to said person. Furthermore, thisinvention relates to pharmaceutical preparations, food additives andfood products comprising hydroxymatairesinol.

BACKGROUND OF THE INVENTION

[0002] The publications and other materials used herein to illuminatethe background of the invention, and in particular, cases to provideadditional details respecting the practice, are incorporated byreference.

[0003] Lignans are defined as a class of phenolic compounds possessing a2,3-dibenzylbutane skeleton. They are formed by coupling of monomericunits called precursors such as cinnamic acid, caffeic, ferulic,coumaric, and gallic acids (Ayres and Loike, 1990). Lignans are widelydistributed in plants. They can be found in different parts (roots,leafs, stem, seeds, fruits) but mainly in small amounts. In many sources(seeds, fruits) lignans are found as glycosidic conjugates associatedwith fiber component of plants. The most common dietary sources ofmammalian lignan precursors are unrefined grain products. The highestconcentrations in edible plants have been found in flaxseed, followed byunrefined grain products, particularly rye. Mammalian lignan productionfrom different plant food are given in Table 1.

[0004] Considerable amounts of lignans are also found in coniferoustrees. The type of lignans differs in different species and the amountsof lignans vary in different parts of the trees. The typical lignans inheart wood of spruce (Picea abies) are hydroxymatairesinol (HMR),(α-conidendrin, conidendrinic acid, matairesinol, isolariciresinol,secoisolariciresinol, liovile, picearesinol, lariciresinol andpinoresinol (Ekman 1979). The far most abundant single component oflignans in spruce is HMR, about 60 percent of total lignans, whichoccurs mainly in unconjugated free form. Lignan concentration in thickroots is 2-3 percent. Abundance of lignans occur in the heart wood ofbranches (5-10 percent) and twists and especially in the knots, wherethe amount of lignans may be higher than 10 percent (Ekman, 1976 and1979). These concentrations are about hundred-fold compared to groundflax powder known as lignan-rich material.

[0005] The chemical structure of hydroxymatairesinol is

[0006] Lignans can be isolated e.g. from compression-wood fiber. Thesefibers originate from compression wood of stems and knots (oversize chipfraction) worsen the quality of paper (Ekman, 1976).

[0007] Plant lignans such as matairesinol and secoisolariciresinol, areconverted by gut microflora to mammalian lignans, enterolactone andenterodiol, correspondingly (Axelson et al. 1982). They undergo anenterohepatic circulation and are excreted in the urine as glucuronideconjugates (Axelson and Setchell, 1981). As an experimental evidence forthe chemopreventive actions of lignans, supplementation of a high-fatdiet with lignan-rich flaxseed flour (5% or 10%) or flaxseed lignans(secoisolariciresinol-diglycoside, SDG) prevented the development ofantiestrogen-sensitive DMBA-induced breast cancer in the rat (Serrainoand Thompson 1991 and 1992; Thompson et al. 1996a and 1996b). Theyreduced the epithelial cell proliferation, nuclear aberrations, thegrowth of tumors, and the development of new tumors. High lignan intakemay also protect against experimental prostate and colon cancers.Dietary rye (containing lignans), prevented at early stages the growthof transplanted Dunning R3327 prostatic adenocarcinomas in rats (Zhanget al. 1997; Landström et al. 1998). The percentage of animals bearingpalpable tumors, the tumor volume, and the growth rate weresignificantly lower. Further, flaxseed or SDG supplementation inhibitedthe formation of chemically induced aberrant crypts in rat colon(Serraino and Thompson 1992; Jenab and Thompson 1996). The antitumoraction may therefore be due to weak estrogen-antiestrogen-likeproperties and/or other mechanisms, which are not well understood.

[0008] Urinary excretion and serum concentrations of enterolactone arelow in women diagnosed with breast cancer (Ingram et al. 1997; Hultén etal. 1998) suggesting that lignans are chemopreventive. Mammalian lignans(enterolactone and enterodiol) have been hypothesized to modulatehormone-related cancers, such as breast cancer, because of theirstructural similarities to the estrogens. Enterolactone had weakestrogenic potency in MCF-7 cells (Mousavi and Adlercreutz 1992), buthad no estrogenic response in mouse uterine weight (Setchell et al.1981). As a sign of estrogen-like activity, SDG feeding during pregnancyand lactation to rats increased the uterine weight at weaning but theeffect was not evident at later stages (Tou et al. 1998). Possibleantitumor effects have also been associated with their antiestrogenicactions (Waters and Knowler, 1982). The inhibition of aromatase bymammalian lignan, enterolactone, would suggest a mechanism by whichconsumption of lignan-rich plant food might contribute to reduction ofestrogen-dependent diseases, such as breast cancer (Adlercreutz et al.1993, Wang et al. 1994). The potential antioxidant activity of lignanscould also represent a mechanism associated with the preventive actionof lignans in the development of cancers. Further, mammalian lignanshave shown to inhibit the conversion of testosterone to5α-dihydrotestosterone (DHT), the potent intracellular androgen, at theconcentrations which are achievable in humans (Evans et al. 1995). Thereduction in DHT concentration would modify the risk of prostate cancer(PC) and benign prostatic hyperplasia (BPH).

[0009] It is possible that lignans as precursors of enterolactone couldalso alleviate lower urinary tract symptoms (LUTS) and gynecomastia. Onthe basis of the results obtained in the animal model, we have suggestedthat estrogens play an essential role in the development of the musculardysfunction involved in urethral dyssynergia seen as bladder neckdyssynergia or external sphincter pseudodyssynergia (Streng et al.unpublished observations). Such neuromuscular changes are at leastpartially reversed by an aromatase inhibitor (MPV-2213ad) indicating therole of estrogens. Further, gynecomastia, which is induced by exposureto estrogens or in the presence of increased ratio of estrogen toandrogens. Gynecomastia can be successfully treated with an aromataseinhibitor. The capability of lignans to inhibit 5α-reductase and/oraromatase combined with their potential antioxidant activity mayrepresent mechanisms associated with the preventive action of lignans inthe development of hormone-related diseases in male organism.

[0010] No data is available on the possible effects of lignans inhumans. The current theories about lignan action in humans have beenderived from studies on the effects of diets supplemented with flaxseedproducts (and thus lignans). Flaxseed in human female diet causedchanges in menstrual cycle (Phipps et al. 1993). The subjects, allnormally cycling women, showed a longer mean length of luteal phase andhigher progesterone/17β-estradiol ration in serum during the lutealphase when they took 10 g of flax seed powder/day in addition to theirhabitual diets (Phipps et al. 1993). No significant differences betweenflax and control cycles or concentrations of either estrone or17β-estradiol were found. Neither there were any significant differencesbetween flax and control groups for concentrations of serum estrogens inpostmenopausal women (Brzezinski et al. 1997). Flaxseed supplementationincreased SHBG (protein which binds estradiol with high capacity)concentration in serum. This is a typical estrogenic effect in the livertissue. Increased SHBG concentration on the other hand reducesbioavailability of endogenous estrogens. In healthy young men, theshort-term (6 weeks) flaxseed supplementation of the diet (10 g/d inmuffins) had no significant effect on plasma testosterone concentrations(Shultz et al. 1991) indicating a lack of estrogenicity in the maleorganism. All together, these studies indicate that lignans may haveweak hormonal (estrogenic and antiestrogenic) effects, but the mechanismof their action cannot be fully described by the hormonal effects.

[0011] In conclusion, isolated mammalian lignans have not been availableearlier in sufficient amounts to be used in animal experiments orclinical trials, and the only possibility to increase lignan intake hasbeen to increase the consumption of fiber-rich food items such asflaxseed. HMR or any other lignan that is efficiently converted toenterolactone, and can be produced/isolated in large quantities would bevaluable in the development of pharmaceutical preparations and foodproducts such as functional foods for chemoprevention of cancer andother hormone-related diseases and cardiovascular diseases.

SUMMARY OF THE INVENTION

[0012] According to one aspect, this invention concerns a method forprevention of a cancer, a certain non-cancer, hormone dependent diseaseand/or a cardiovascular disease in a person comprising administering tosaid person an effective amount of hydroxymatairesinol, a geometricisomer or a stereoisomer thereof, or a salt thereof.

[0013] According to an other aspect, the invention concerns a method forincreasing the level of enterolactone or another metabolite ofhydroxymatairesinol in a person's serum thereby causing prevention of acancer or a certain non-cancer, hormone dependent disease in a personcomprising administering to said person an effective amount ofhydroxymatairesinol, a geometric isomer or a stereoisomer thereof, or asalt thereof.

[0014] According to a third aspect, the invention concerns apharmaceutical preparation comprising an effective amount ofhydroxymatairesinol, a geometric isomer or a stereoisomer thereof, or apharmaceutically acceptable salt thereof, in combination with apharmaceutically acceptable carrier.

[0015] According to a fourth aspect, the invention concerns a productcomprising hydroxymatairesinol, a geometric isomer or a stereoisomerthereof, or a salt thereof, in combination with a solid or liquidcarrier, for use as additive to a food product.

[0016] According to a fifth aspect, the invention concerns a foodproduct comprising an effective amount of hydroxymatairesinol, ageometric isomer or a stereoisomer thereof, or a salt thereof.

[0017] According to still one aspect, the invention concerns a methodfor increasing the stability of a food product comprising the additionto said food product of an effective amount of hydroxymatairesinol, ageometric isomer or a stereoisomer thereof, or a salt thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIG. 1 shows the concentration-related inhibition of aromatase bylignans in JEG-3 cells.

[0019]FIG. 2 shows the proliferation of MCF-7 cells in the presence andabsence of HMR.

[0020]FIG. 3 shows the uterine wet weight of immature rats treated withHMR or with an aromatase inhibitor.

[0021]FIG. 4 shows the antitumor activity of HMR against DMBA-inducedmammary gland tumors in female rats.

[0022]FIG. 5 shows the excretion of enterolactone in the urine of ratstreated with different doses of HMR.

DETAILED DESCRIPTION OF THE INVENTION

[0023] This invention relates to the use of a lignan,hydroxymatairesinol (HMR), for the prevention of cancer, non-cancer,hormone dependent diseases and cardiovascular diseases by adding saidHMR into food or by using it as a pharmaceutical preparation.Surprisingly, HMR is metabolized in vivo to enterolactone, which isassumed to account at least partly for the antitumor properties of thelignans. Antioxidative activity of HMR in vitro is strong and thisproperty indicates that HMR can also prevent cardiovascular diseasesthrough the protective effect against damaging free oxygen species inthe body. The invention relates also to the use of HMR as a foodadditive to increase the food stability (i.e. inhibit lipid and pigmentoxidations and vitamin losses which cause loss of nutritional value anddevelopment of off-flavors in food).

[0024] The method according to this invention is particularly effectivein the prevention of cancers such as breast cancer, prostate cancer andcolon cancer, non-cancer, hormonal dependent diseases such as lowerurinary tract symptoms, urethral dyssynergia, bladder instability,bladder outlet obstruction, benign prostatic hyperplasia, andgynecomastia in men, and cardiovascular diseases resulting from oxidizedLDL in serum.

[0025] The pharmaceutical preparation according to this invention ispreferably an oral formulation. The required amount of the activecompound (HMR) will vary with the particular condition to be prevented.A typical dose ranges from about 10 to about 100 mg per day and adultperson. Typical dosage forms include, but are not limited to, oraldosage forms such as powders, granules, capsules, tablets, caplets,lozenges, liquids, elixirs, emulsions and suspensions. All such dosageforms may include conventional carriers, diluents, excipients, bindersand additives known to those skilled in the medicinal and pharmaceuticalarts.

[0026] The pharmaceutical carriers typically employed may be solid orliquid. Thus, for example, solid carriers include polysaccarides such aslactose, sucrose, gelatin, agar, while liquid carriers include aqueoussolutions of salts, polysaccarides, complexing agents, surfactants,syrups, vegetable oils such as peanut oil or olive oil, and certainalcohols. However, any pharmaceutically acceptable solid or liquidcarrier can be used in the pharmaceutical preparation according to theinvention, except that the formulation cannot be a mixture of only theactive agent and plain water.

[0027] In the food additive of the invention, the carrier to be used,can be any non-toxic solid or liquid carrier acceptable for use in foodand suitable to be admixed with HMR without affecting the properties ofHMR. The role of the material is mainly to make the exact dosage of HMReasier. A suitable concentration is for example 100 mg to 1 g of HMR per100 g of carrier. Typical dosage forms include, but are not limited to,oral dosage forms such as powders, granules, capsules, tablets, caplets,lozenges, liquids, elixirs, emulsions and suspensions. All such dosageforms may include conventional carriers, diluents, excipients, bindersand additives known to those skilled in the art.

[0028] Typical solid carriers include polysaccarides such as lactose,sucrose, gelatin, agar, while liquid carriers include aqueous solutionsof salts, polysaccarides, complexing agents, surfactants, syrups,vegetable oils such as peanut oil or olive oil, and certain alcohols.However, any acceptable solid or liquid carrier can be used in the foodadditive according to the invention, except that the food additiveformulation cannot be a mixture of only the active agent and plainwater.

[0029] The food product according to this invention is especially afunctional food, a nutritional supplement, a nutrient, a pharmafood, anutraceutical, a health food, a designer food or any food product. Asuitable concentration of HMR in the food product is, for example, 1 to20 mg of HMR per 100 g of food product.

[0030] The functional food according to this invention can, for examplebe in the form of butter, margarin, biscuits, bread, cake, candy,confectionery, yogurt or an other fermented milk product, or cereal suchas muesli.

[0031] The addition of hydroxymatairesinol is particularly useful toincrease food stability in the meaning of inhibitition of lipid, vitaminand pigment oxidations, which cause loss of nutritional value anddevelopment of off-flavors in food. A suitable concentration of HMR forthis purpose is, for example, about 0.1%.

[0032] Isolation of HMR for use in this invention can be made fromoversize chip fraction (containing branches, twists and knots) ofcompression wood. According to a preferred embodiment, thehydroxymatairesinol is derived from coniferous wood. It has been shownthat hydroxymatairesinol is the dominant lignin in coniferous wood. Forexample, in knotrich spruce wood 85% or even more of the lignins ishydroxymatairesinol.

[0033] The properties of HMR were studied by seven different assays:

[0034] 1. Measurement of antioxidant capacity in vitro

[0035] 2. Measurement of aromatase inhibiting capacity in JEG-3 cells

[0036] 3. Measurement of estrogenic and antiestrogenic activity in MCF-7cell cultures

[0037] 4. Evaluation of estrogenic and antiestrogenic activity byuterine growth bioassay

[0038] 5. Measurement of estrogenic and antiestrogenic activity in adultmale rats

[0039] 6. Investigating the antitumor activity in rat DMBA-inducedmammary cancer model

[0040] 7. Analysis of metabolites from rat urine after different dosesof HMR

[0041] The isolation and purification of HMR in sufficient amounts forbiological tests has been impossible earlier because it is a componentof wood lignans, which have been relatively poorly characterized.Understanding the distribution of HMR in different parts of spruce(Ekman 1976 and 1979) has given the opportunity to study lignans andespecially HMR in detail.

[0042] A linear correlation was found between the doses of HMR and theamounts of urinary enterolactone. Enterolactone is a well knownmammalian lignan formed by intestinal bacteria from matairesinol or byoxidation of enterodiol (Axelson and Setchell 1981; Axelson et al.1982). Only minute amounts of unmetabolized HMR and other metabolites(enterodiol and 7-hydroxyenterolactone) were found in urine. Theiramounts remained unchanged when the daily dose of HMR was increased.These findings suggest that HMR was metabolized to enterolactone, and,further, enterolactone derived from HMR through demethylation anddehydroxylation steps is not converted to enterodiol. Based on thestructure of HMR one had expected that 7-hydroxyenterolactone were themain metabolite of HMR, but this was not the case. This hydroxyl groupis eliminated in the metabolism. The metabolism of HMR differs from thatof SDG. SDG is metabolized to enterodiol which is partly oxidized toenterolactone (Rickard et al. 1996; Lampe et al. 1994). HMR thus offersan advantage over SDG as a direct precursor of enterolactone. HMR hadweak if any estrogenic action in rat uterus or in the male organism. Itexerted weak but not significant estrogen-like activity in MCF-7 cells.No antiestrogenic activity was demonstrated for HMR. Therefore, it issurprising that it had highly significant antitumor activity inDMBA-induced tumor model in rats as shown in FIG. 2. The activity of HMRmay be due to HMR itself or to enterolactone. However, nodose-dependence was found in the chemopreventive action of HMR whengiven in two different doses (3 and 15 mg/kg) to rats afterDMBA-treatment. Thus HMR needs not to be converted to enterolactone tohave an antitumor effect or smaller doses of these lignans aresufficient to accomplish the maximal chemopreventive effects.

[0043] HMR is very effective antioxidant as shown in Tables 2 and 3. Itis one of the most potent known inhibitors of lipid peroxidation andexcellent inhibitor of LDL oxidation. Inhibition of LDL oxidation isconsidered to be of special importance in humans as the concentration ofoxidized LDL in serum is considered to be one of the best predictors ofcardiovascular diseases such as atherosclerosis. HMR may serve as a foodadditive to increase the food stability (i.e. inhibit vitamin, lipid andpigment oxidations which cause loss of nutritional value and developmentof off-flavors in food), because HMR was much better superoxide anionscavenging and peroxyl radical scavenging agent than well knownantioxidants butylated hydroxyanisol (BHA) and butylated hydroxytoluene(BHT), which are commonly used for increasing the food stability.

EXPERIMENTS Chemicals

[0044] Various lignans were tested in vitro for their estrogenicity,antiestrogenicity, capability to inhibit aromatization and for theirantioxidative properties. The test compounds were purchased from thefollowing sources: enterodiol and enterolactone from Plantech, London,UK, and 7-hydroxyenterolactone containing two 7-OH enantiomers was agenerous gift from Dr. Kristina Wähälä, Department of Applied Chemistry,University of Helsinki, Finland.

Extraction of HMR from wood

[0045] HMR extracts were isolated from Norway spruce (Picea abies) asdescribed by Ekman, 1976 and Ekman 1979. Shortly, freeze-dryed groundheartwood was Soxhlet-extracted in with hexane to remove non-polarlipophilic extractive. The wood sample was re-extracted in the sameapparatus with acetone/water (9:1 v/v) to give crude lignans.Hydroxymatairesinol (HMR) and its isomer were isolated andre-chromatographed with XAD-resin for further purification.

Measurement of antioxidant capacity in vitro

[0046] The antioxidative capacity of lignans was estimated by fourdifferent methods: 1) inhibition of lipid peroxidation, 2) inhibition oflow density lipoprotein (LDL) oxidation, 3) superoxide anion scavengingand 4) peroxyl radical scavenging assays.

[0047] Inhibiton of lipid peroxidation was evaluated on the basis oftheir potency to inhibit tert-butylhydroperoxide-induced lipidperoxidation (t-BuOOH-LP) in rat liver microsomes in vitro (Ahotupa etal. 1997). The test for the t-BuOOH-LP was carried out as follows: Thebuffer (50 mM sodium carbonate, pH 10.2, with 0.1 mM EDTA) was pipettedin a volume of 0.8 ml in the luminometer cuvette. Twenty microliters ofdiluted liver microsomes, final concentration 1.5 mg protein/ml, wasadded, followed by 6 ml of lumninol (0.5 mg/ml) and test chemicals. Thetest compounds were added to incubation mixtures in a small volumediluted in ethanol or dimethylsulphoxide (2% of incubation volume), andthe lipid peroxidation potency was compared to that of the vehicle(ethanol or dimethyl sulphoxide). The reaction was initiated by 0.05 mlof 0.9 mM t-BuOOH at 33° C. Chemiluminescence was measured for about 45min at 1 min cycles, and the area under curve (integral) was calculated.Chemiluminescence measurements were carried out using a Bio-Orbit 1251Luminometer (Bio-Orbit, Turku, Finland) connected to a personal computerusing dedicated software for the assays.

[0048] Inhibition of LDL oxidation was estimated as described by Ahotupaet al, 1996. Shortly: LDL was isolated by precipitation with bufferedheparin. After resuspendation in phosphate buffer, 20 mM CuCl₂ was addedand the mixture was incubated for 3 hrs at +37° C. After this, LDLlipids were extracted with chloroform-methanol, dried under nitrogen,redissolved in cyclohexane and analyzed spectrophotometrically at 234mn. The intensity of absorbance is indicative of LDL oxidation. To testthe ability of different compounds to prevent LDL oxidation, thecompounds were added to the incubation mixture prior addition of CuCl₂.Possible interference of test compounds with the assay procedure wasexcluded by measuring the absorption at 234 nm before and after theincubation period. For those compounds which showed antioxidativepotency at the starting concentration (0,1 mM), IC-50 values (i.e.concentrations at which test compound inhibited LDL oxidation by 50%)was determined.

[0049] Superoxide anion scavenging method was based on the superoxideanion produced in controlled conditions by xanthine-xanthine oxidasesystem and detection of the generated reactive oxygen species byluminometer (Ahotupa et al., 1997). The ability of test compounds todecrease the chemiluminescence was evaluated. IC-50 concentration(concentration which prevented the chemiluminescence by 50%) wascalculated.

[0050] Peroxyl radiocal scavenging assay was based on generation ofperoxyl radicals by thermal decomposition of2,2′-azobis(2-amidinopropane)HCl and their detection bychemiluminescence (Ahotupa et al., 1997). The results were calculated asthe stochiometric factor, i.e. how many moles of peroxyl radicals can bescavenged by one mole of the test compound.

Measurement of aromatase inhibiting capacity in JEG-3 cells

[0051] The effects of HMR and structurally related lignans(enterolactone, enterodiol and 7-hydroxyenterolactone) were studied onformation of ³H-17β-estradiol from ³H-andostenedione in JEG-3 cells,human choriocarcinoma cell line. The JEG-3 choriocarcinoma cells are auseful aromatase model enabling the study of aromatase inhibition invitro (Krekels et al 1991). Cells were maintained in DMEM containing 10%fetal calf serum (FCS). The incubation mixture contained 50 μl³H-androst-4-ene, 3,17-dione (0.5 nM), 50 μl unlabelled androstenedione(0.5 nM), 100 μl test compounds (10 mM) and 800 μl cell suspension (1million cells). After the incubation for 4 h, unlabelled carriers(androstenedione, testosterone, 17β-estradiol and estrone) were added.The steroids were extracted twice with 3.0 ml dichloromethane. HPLC wasused for separation and quantification of the radiolabelled³H-17β-estradiol as previously described (Mäkelä et al. 1995). Thecolumn system consisted of a guard column followed by a C18 150×3.9 mmID analytical column (Technopak 10 C18 HPLC Technology; WellingtonHouse, Cheshire, UK). The mobile phase was acetonitrile/water (35/65)and the flow rate was 1.2 ml/min. For in-line detection of theradioactive metabolites, the eluent of the HPCL column was continuouslymixed with liquid scintillant and then monitored with in-lineradioactivity detector.

Measurement of estrogenic and antiestrogenic activity in MCF-7 cellcultures

[0052] The MCF-7 cell line (human breast cancer cells) stock cultureswere grown in phenol red free RPMI medium supplemented with 5% FCS, 100U/ml penicillin and 100 μg/ml streptomycin, 10 μg/ml insulin and 1 nM17β-estradiol in T-75 cell culture bottles. The medium was replaced withfresh ones three times per week. The stock cultures were harvested bytrypsinization and suspended in 10 ml phenol red free versene solutionand centrifuged for 5 min 800 rpm. The cell pellet was carefullyresuspended into RPMI medium supplemented with 5% dextran charcoalstripped FCS (dcFCS) and seeded on 6 well plates 50,000 cells/3.0 mlmedium/well. On the second day of culture the medium was changed andtest compounds were added. To test the estrogenicity of the lignancompounds, they were diluted in ethanol and added to cell cultures infinal concentration of 1.0 M. In each proliferation assay 1.0 nM17β-estradiol solution in ethanol was used as a positive control forestrogenic response. Equal amounts of ethanol were added to controlwells. To test the antiestrogenicity both 17β-estradiol and lignansolutions were added to cell cultures. The cells were cultured for 5 to7 days in the presence of test compounds, and the medium was changedevery second day. Cell proliferation was quantified by counting thereleased nuclei with Coulter counter.

Evaluation of estrogenic and antiestrogenic activity in immature ratuterotropic test

[0053] The estrogenicity HMR was evaluated by the uterotropic assay inimmature rats which was performed as described earlier (Jordan et al.1977), with the exception of treatment time which was 7 days instead of3 days in the reference study. The treatment time was longer because ofthe expected weak estrogenicity of the test compound. The treatment ofimmature rats with an aromatase inhibitor (MPV-2213ad), which preventsbiosynthesis of estradiol, was used as a methodological control fornon-estrogen-stimulated uterus.

Evaluation of estrogenic and antiestrogenic activity in adult male rats

[0054] Estrogenic (antiandrogenic) and antiestrogenic effects of HMRwere studied in intact and hypoandrogenic Noble strain male rats (age6-9 month), correspondingly. The chronic hypoandrogenic state with bothstructural and functional changes in the male reproductive tract wasinduced by neonatal estrogenization (diethylstilbestrol, 10.0 μg/kg bodyweight in rape oil s.c. on postnatal days 1-5). These changes are knownto be partly reversible by aromatase inhibitor treatment consistingdaily dose of MPV-2213 ad 10-30 mg/kg body weight (Streng et al.unpublished observations).

[0055] Animals were fed the soy-free basal diet (SDS, Whitham Essex,England) and they had a free access to water. Twelve of both intact andhypoandrogenic animals were cavaged in daily dosage of HMR 50 mg/kg bodyweight in rape oil. Another twelve animals from both animal models werecavaged with rape oil only as a placebo treatment. After four-weektreatment the animals were sacrificed. The weights of testis andaccessory sex glands (ventral prostate, seminal vesicles and coagulatinggland) were measured. Serum and testis testosterone and pituitary andserum luteinizing hormone (LH) levels were measured by immunoassays(Haavisto et al. 1993).

Investigating the antitumor activity in rat DMBA-induced mammary cancermodel

[0056] Antitumor activity of HMR in rat mammary cancer was studied asdescribed earlier (Kangas et al. 1986). Fifty-day-old femaleSprague-Dawley rats were given 12.0 mg DMBA (dimethylbentz[a]anthracene)by cavage. After approximately 6 weeks palpable tumors could bedetected, whereafter the width (w) and the length (1) of the tumors weremeasured once a week to determine the tumor volumes according to aformula V =(πw² 1)/12. The rats were also weighed once a week. The ratswere allocated in 3 different groups so that the total number of tumorsin the beginning of the experiment was similar in each group: (1)Control group 8 animals, (2) HMR 3.0 mg/kg 7 animals, and (3) HMR 15.0mg/kg 7 animals, one of which had to be killed before the end of theexperiment.

[0057] HMR was given per os starting 9 weeks after the DMBA-induction,i.e. 3 weeks after palpable tumors appeared, and was given daily for 7.5weeks. At the end of the experiment the tumors were classified in groupsaccording to their growth pattern: 1. Growing tumors (PD =progressivedisease); 2. Non-growing, stabilized tumors (SD =stabilized disease, nochange in tumor volume or regression less than 75%; 3. Regressing tumors(PR =partial response, regression of tumor volume more than 75%); 4.Disappeared tumors (CR =complete response, no palpable tumor).

Analysis of metabolites from rat urine receiving different doses of HMR

[0058] Ten Sprague-Dawley male rats (age 4 month) were used to study themetabolism of HMR in vivo. Animals were housed in pairs with 12 hlight:dark cycle and had free access to water and soy-free basal diet(SDS, Whitham Essex, England) during the metabolism study.

[0059] Rats were cavaged with HMR dissolved in 10% ethanol in PEG indoses 3, 15, 25 and 50 mg/kg body weight once a day for two days. Aftersecond cavaging the 24 hour urine was collected in metabolic cages incollection jars containing 120 μl 0,56 M ascorbic acid and 120 μl 0,15 MNa-azide as preservatives. The centrifuged urine volumes were measuredand stored in −20° C. For pretreatment 750 μl 0.2 M acetate buffer (pH4.0±0.1) was added to 3.0 ml thawed urine aliquots. Sep-Pak C18 columns(100 mg silica based resin/column) were used for urine extractions.Columns were preconditioned with 3.0 ml H₂O, 3.0 ml methanol and 3.0 mlacetate buffer. After urine had filtered through the column and washedwith 3.0 ml of acetate buffer polyphenolics were eluted with 3.0 mlmethanol. The eluate was evaporated to dryness under nitrogen in +45° C.water bath and dryed residues were redissolved in 3.0 ml of 0.2 Macetate buffer. 30 μl Helix pomatia enzyme mix was added and thesolutions were incubated in +37° C. to hydrolyze both glucuronides andsulfates. 300 μl of flavone stock solution (100 μg/ml in EtOH) was addedinto hydrolyzed samples. The samples were extracted in C-18 columns andevaporated to dryness as described above and stored in −20° C. untilanalyzed with GC-MS.

[0060] The evaporated urine samples were dissolved in pyridine, andsilylated by adding BSTFA:TMCS (10:1) silylation reagent. The GC-MSanalyses of the silylated samples were performed with an HP 6890-5973GC-MS instrument. The GC column was an HP-1 crosslinked methylpolysiloxane column (15 m×0,25 mm i.d., 0,25 μm film thickness). Heliumwas used as carrier gas at a flow of 1 ml/min. The GC-oven wastemperature programmed from 60° C. to 290° C., at 8° C./min heatingrate. The GC-injector was set in split-mode at a split ratio of 1:15.The injector temperature was 250° C. Compound identifications were basedon mass spectra. The quantitative calculations were based on uncorrectedpeak areas of target components relative to the internal standard.

RESULTS Assessment of antioxidant capacity in vitro

[0061] HMR had stronger lipid peroxidation capacity than any otherlignan or flavonoid in our tests (Table 2). HMR was compared to wellknown antioxidants TROLOX, which is a water soluble vitamin Ederivative, and BHA and BHT in the ability of inhibiting lipidperoxidation, inhibition of LDL oxidation, and scavenging superoxide andperoxyl radicals (Table 3). HMR was as a whole the strongestantioxidant, more effective than BHA or BHT in all assays, and strongerthan TROLOX in all assays except for lipid peroxidation inhibitionassay, where the compounds were almost equally active.

Aromatase inhibiting capacity in JEG-3 cells

[0062] The inhibition of ³H-17β-estradiol formation from³H-androstenedione in JEG-3 cells was tested at different concentrationsof HMR. The inhibitory capacity of HMR was compared to enterolactone,7-hydroxyenterolactone and enterodiol. Enterolactone caused adose-dependent inhibition of aromatization within the concentrationrange of 1.0 to 10.0 μM. It was further shown that enterodiol wasnoninhibitory indicating that the lactone ring is critical for theinhibition. 7-hydroxyenterolactone and hydroxymatairesinol had noinhibitory effects (FIG. 1) indicating the importance of the number andlocation hydroxyl groups in the lignan molecule for the aromataseinhibition.

Estrogenic and antiestogenic activity in MCF-7 cell cultures

[0063] HMR had very weak, not statistically significant estrogenic orantiestrogenic activity in MCF-7 cell proliferation assays as shown inFIG. 2.

Evaluation of estrogenic and antiestrogenic activity in immature ratuterotropic test

[0064]FIG. 3 illustrates the effects of HMR on the uterine growth of theimmature rats. HMR had no significant estrogenic effect on the uterineweight gain of the immature rats. Neither did HMR reduce the weightgains indicating no antiestrogenic effect. Aromatase inhibitor preventedthe increase of uterine weight, as expected, indicating that the methodfor the measurement of the aromatase inhibitors was adequate.

Evaluation of estrogenic and antiestrogenic activity in adult male rats

[0065] After a 4-week treatment with HMR, no significant changes in theweights of accessory sex glands and testis were observed in control andhypoandrogenic animals (Table 4). There were no significant changes intestosterone or LH concentrations, either (Table 5). These resultsindicate, that HMR is not a full estrogen agonist in male organism,because it does not exert the typical estrogenic activity onhypothalamus-hypophysis-gonad-axis (inhibition of LH and androgensecretion). Neither is HMR an antiestrogen because it does not reversethe changes induced by neonatal estrogenization in the male rat.

[0066] Investigating the antitumor activity in rat DMBA induced mammarycancer model

[0067] Number of growing (PD) versus stable (SD) tumors, regressing (PR)tumors and disappeared (CR) tumors is presented in FIG. 4. The antitumoreffect of HMR was found to be statistically very significant. There wasno clear dose-dependency of antitumor action in this model. Bothantioxidative and tumor growth regressing properties of HMR maytherefore be connected with the in vivo antitumor activity. Themechanism of antitumor activity of HMR in vivo is still unknown.

Analysis of metabolites from rat urine after different doses of HMR

[0068]FIG. 5 illustrates that the main excreting metabolite of HMR inrats is enterolactone, which may be the biologically active compound.This is surprising taking into account the chemical structure of HMR,because one would expect hydroxyenterolactone to be the main metabolite.The metabolism of HMR to enterolactone may be catalyzed by bacterialintestinal flora rather than by the rat liver.

CONCLUSIONS

[0069] Hydroxymatairesinol (HMR) has antitumor activity either asunchanged compound and/or after conversion to enterolactone in DMBAinduced breast cancer model. HMR has therefore a potential to havebeneficial effects in humans who are at risk of developing breast cancer(BC), prostate cancer (PC), colon cancer or benign prostatic hyperplasia(BPH). HMR is metabolized to enterolactone which inhibits aromatizationin vitro. HMR may as a precursor of aromatase inhibitor also prevent thedevelopment of lower urinary tract symptoms (LUTS), bladder instability,bladder outlet obstruction, urethral dyssynergia, and gynecomastia. HMRhas also strong antioxidative activity and may therefore be used as foodadditive (antioxidant). HMR as pharmaceutical product or dietarysupplement may have advantageous cardiovascular effects in humans.Addition of HMR to food to make innovative new functional food,nutraceutical, health food, pharmafood, designer food or novel food isfeasible.

[0070] It will be appreciated that the methods of the present inventioncan be incorporated in the form of a variety of embodiments, only a fewof which are disclosed herein. It will be apparent for the specialist inthe field that other embodiments exist and do not depart from the spiritof the invention. Thus, the described embodiments are illustrative andshould not be construed as restrictive. TABLE 1 Production of mammalianlignans from different plant food by in vitro fermentation with humanfecal flora. μg/100 g FLAXSEED FLOUR 68 000   SOYBEAN 170 CEREAL BRANS:WHEAT 570 OAT 650 WHOLE CEREALS: RYE 160 POTATO  80 CARROT 350 ONION 110

[0071] TABLE 2 ANTIOXIDANT PROPERTIES OF LIGNANS AND SOME RELATEDFLAVONOIDS IN VITRO BY LIPID PEROXIDATION INHIBITION TEST. Antioxidativecapacity (t-BuOOH-LP) Compound IC₅₀ (μM) Flavonoids kaempferol 0.9(3,4′,5,7-tetrahydroxy- flavone) quercetin 0.4(3,3′,4′,5,7-pentahydroxy- flavone) kaempferide 0.5(3,5,7-trihydroxy-4′-methoxy- flavone) Lignans enterolactone 15.92,3-bis-(3′-hydroxybenzyl)-butyrolactone enterodiol 12.72,3-bis-(3′-hydroxybenzyl)-butane-1,4-diol hydroxymatairesinol 0.08

[0072] TABLE 3 COMPARISON OF ANTIOXIDATIVE EFFECTS OF HMR AND KNOWNANTIOXIDANTS IN VITRO. IC-50 concentrations have been presented, exceptfor peroxyl radical scavenging assay where the stochiometric factor(i.e. how many moles of peroxyl radical one mole of test compound canscavenge). HMR¹ TROLOX² BHA³ BHT⁴ Inhibition of lipid 0.06 μM 0.02 μM1.1 μM 15.3 μM peroxidation Inhibition of LDL 2.0 μM 2.7 μM notdetermined oxidation Superoxide anion 5.6 μM 25 μM 15 μM >1 mMscavenging Peroxyl radical 1:4 1:2 not determined scavenging

[0073] TABLE 4 The effect of four week exposure to HMR on male ratreproductive organ relative weights¹ mg/kg body weight Body weightVentral Seminal Coaculating Treatment n g Testis Prostate vesicle glandIntact animals Placebo 12 426 ± 28 4362 ± 170 909 ± 146 412 ± 43 223 ±49  HMR 12 447 ± 38 4223 ± 304 938 ± 148 419 ± 59 204 ± 48  50 mg/kgHypoandrogenemic Placebo 12 481 ± 29 3340 ± 509 333 ± 188 249 ± 63 69 ±49 animals HMR 12 455 ± 36 3276 ± 327 378 ± 198 266 ± 49 70 ± 30 50mg/kg

[0074] TABLE 5 The effect of four week exposure to HMR on male rattestosterone and LH concentrations¹ Testis Serum testosteronetestosterone Pituitary LH Serum LH Treatment n (ng/testis) (ng/ml)(μg/pit) (ng/ml) Intact animals Placebo 12 97,6 ± 46,3 2,405 ± 1,1226,747 ± 2,479 1,804 ± 1,294 50 mg/kg HMR 12 112,9 ± 58,5  2,770 ± 1,4216,838 ± 2,061 1,088 ± 0,352 Hypoandrogenic Placebo 12 63,5 ± 25,9 1,197± 0,663 8,673 ± 2,224 0,712 ± 0,371 animals 50 mg/kg HMR 12 48,0 ± 15,20,939 ± 0,431 7,530 ± 2,286 0,854 ± 0,333

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1. A pharmaceutical preparation comprising an effective amount of anactive agent, which is hydroxymatairesinol, a geometric isomer or astereoisomer thereof, or a pharmaceutically acceptable salt thereof, incombination with a pharmaceutically acceptable carrier, wherein saidagent is either the sole lignan present or the most abundant singlecomponent of a lignan mixture, and wherein said carrier is a solidcarrier or a liquid carrier, provided that said pharmaceuticalpreparation is other than a mixture of only the active agent and plainwater.
 2. The pharmaceutical preparation according to claim 1, whereinsaid agent has been derived from coniferous wood.
 3. A productcomprising an active agent, which is hydroxymatairesinol, a geometricisomer or a stereoisomer thereof or an acceptable salt thereof, incombination with an acceptable carrier, for use as additive to a foodproduct, wherein said agent is either the sole lignan present or themost abundant single component of a lignan mixture, and wherein saidcarrier is a solid carrier or a liquid carrier, provided that theproduct is other than a mixture of only the active agent and plainwater.
 4. The product according to claim 3, wherein said agent has beenderived from coniferous wood.
 5. The product according to claim 3,wherein said food product is selected from the group consisting of afunctional food, a nutritional supplement, a nutrient, a pharmafood, anutraceutical, a health food, a designer food or any food product.
 6. Afood product comprising an effective amount of agent which ishydroxymatairesinol, a geometric isomer or a stereoisomer thereof, or anacceptable salt thereof, wherein said agent has been derived fromconiferous wood.
 7. The food product according to claim 6 wherein saidfood product is selected from the group consisting of a functional food,a nutritional supplement, a nutrient, a pharmafood, a nutraceutical, ahealth food, a designer food or any food product.